U.S. patent application number 12/093610 was filed with the patent office on 2008-12-18 for methods using pores.
Invention is credited to Yann Astier, Hagan Bayley, Orit Braha.
Application Number | 20080311582 12/093610 |
Document ID | / |
Family ID | 35516986 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080311582 |
Kind Code |
A1 |
Bayley; Hagan ; et
al. |
December 18, 2008 |
Methods Using Pores
Abstract
The invention relates to a method of identifying an individual
nucleotide, comprising (a) contacting the nucleotide with a
transmembrane protein pore so that the nucleotide interacts with
the pore and (b) measuring the current passing through the pore
during the interaction and thereby determining the identity of the
nucleotide. The invention also relates to a method of sequencing
nucleic acid sequences and kits related thereto.
Inventors: |
Bayley; Hagan; (Oxford,
GB) ; Astier; Yann; (Oxford, GB) ; Braha;
Orit; (Oxford, GB) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP;FLOOR 30, SUITE 3000
ONE POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
35516986 |
Appl. No.: |
12/093610 |
Filed: |
November 15, 2006 |
PCT Filed: |
November 15, 2006 |
PCT NO: |
PCT/GB2006/004265 |
371 Date: |
July 28, 2008 |
Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 1/6869 20130101;
G01N 33/48721 20130101; C12Q 1/6869 20130101; C12Q 2565/631
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 2005 |
GB |
0523282.2 |
Claims
1. A method of identifying an individual nucleotide, comprising:
(a) contacting the nucleotide with a transmembrane protein pore so
that the nucleotide interacts with the pore; and (b) measuring the
current passing through the pore during the interaction and thereby
determining the identity of the nucleotide.
2. A method according to claim 1, wherein the interaction involves
the nucleotide reversibly binding to the channel of the pore.
3. A method according to claim 1, wherein the pore is
.alpha.-hemolysin as shown in SEQ ID NO: 2 or a variant
thereof.
4. A method according to claim 3, wherein the variant is
(M113R).sub.7.
5. A method according to claim 1, wherein the pore comprises a
molecular adaptor that facilitates the interaction between the
nucleotide and the pore.
6. A method according to claim 5, wherein the molecular adaptor is
a heptakis-6-amino-.beta.-cyclodextrin (am.sub.7-.beta.-CD).
7. A method according to claim 1, wherein the individual nucleotide
is a monophosphate, diphosphate or triphosphate.
8. A method according to claim 1, wherein the individual nucleotide
is a ribonucleotide.
9. A method according to claim 8, further comprising before step
(a) digesting a ribonucleic acid (RNA) sequence to provide the
individual nucleotide.
10. A method according to claim 1, wherein the individual
nucleotide is a deoxyribonucleotide.
11. A method according to claim 10, further comprising before step
(a) digesting a deoxyribonucleic acid (DNA) sequence to provide the
individual nucleotide.
12. A method according to claim 9, wherein more than one of the
individual nucleotides of the RNA or DNA sequence are contacted
with the pore in a sequential manner such that the identity of the
whole or part of the sequence may be determined.
13. Use of a pore to identify an individual nucleotide.
14. A method of sequencing a target nucleic acid sequence,
comprising: (a) digesting an individual nucleotide from one end of
the target sequence using a processive exonuclease; (b) contacting
the nucleotide with a transmembrane protein pore so that the
nucleotide interacts with the pore; (c) measuring the current
passing through the pore during the interaction and thereby
determining the identity of the nucleotide; and (d) repeating steps
(a) to (c) at the same end of the nucleic acid sequence and thereby
determining the sequence of the nucleic acid.
15. A kit for sequencing a nucleic acid, comprising: a
cyclodextrin; and a processive exonuclease.
16. A kit according to claim 15, further comprising a transmembrane
protein pore.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the identification of individual
nucleotides and other phosphate containing moieties using
transmembrane pores. In particular, the invention relates to the
sequencing of target nucleic acids using transmembrane pores.
BACKGROUND OF THE INVENTION
[0002] The current method for sequencing DNA involves a number of
costly reagents such as fluorescent ddXTPs, dXTPs, primers and
polymerase. This method requires sophisticated equipment, which
needs to be operated by a qualified technician. Also, this method
is limited to sequences of less than one thousand nucleotides in
length.
[0003] Other sequencing methods have been considered in order to
reduce cost, simplify the method, and allow sequencing to take
place out of the lab. Cycle extension, polymerase reading,
exonuclease sequencing, and DNA micro-arrays are methods that have
been considered (Braslavsky, I., B. Herbert, et al. (2003), PNAS
100(7): 3960-3964). These methods have been comprehensively
reviewed (Marziali, A. and M. Akeson (2001), Ann. Rev. Biomed. Eng.
3: 195-223).
[0004] One potential method of sequencing DNA is based on threading
a single strand of DNA through a nanopore and identifying its
sequence from the variation in the ionic current flowing through
the pore as the strand is threaded (Kasianowicz, J. J., E. Brandin,
et al. (1996), Proc. Natl. Acad. Sci. 93: 13770-13773). A second
potential approach is exonuclease sequencing (Chan, E. Y. (2005),
Mutat. Res. 573: 13-40). This method involves digesting the DNA one
nucleotide at a time (Dapprich, J. (1999), Cytomet. 36: 163-168;
and Matsuura, S.-I., J. Komatsu, et al. (2001), Nuc. Ac. Res.
29(16): e79) and then identifying each of the released nucleotides.
However, these methods require modification of the DNA before
digestion or modification of the nucleotides once they have been
released from the DNA by exonuclease. The development of
exonuclease sequencing is currently being held back by the
difficulty in identifying the nucleotides at the single molecular
level as they are released by the enzyme. Investigators have tried
to identify the nucleotides using fluorescent labeling with limited
success.
[0005] Stochastic sensing involves placing a nanometer sized pore
in an insulating lipid bilayer membrane and measuring the ionic
transport through the pore. When an analyte interacts with a
binding site within the pore, a change in the ionic current is
detected (Braha, O., B. Walker, et al. (1997), Chem. & Biol. 4:
497-505; and Bayley, H. and P. S. Cremer (2001), Nature 413:
226-230). The extent and duration of the current block resulting
from each binding event can reveal the identity of the analyte. The
frequency of the binding events can reveal the analyte
concentration. Various binding sites can be created within the pore
by way of protein mutation, chemical modification, and by use of
molecular adaptors and carriers (Gu, L.-Q., O. Braha, et al.
(1999), Nature 398: 686-690; and Braha, O., J. Webb, et al. (2005),
Chem. Phys. Chem. 6: 889-892).
SUMMARY OF THE INVENTION
[0006] It has been surprisingly demonstrated that individual
nucleotides can be identified at the single molecule level from
their current amplitude when they interact with a transmembrane
pore. Hence, stochastic sensing may be used to identify individual
nucleotides and to sequence nucleic acid sequences via exonuclease
sequencing.
[0007] Accordingly, the invention provides a method of identifying
an individual nucleotide, comprising: [0008] (a) contacting the
nucleotide with a transmembrane protein pore so that the nucleotide
interacts with the pore; and [0009] (b) measuring the current
passing through the pore during the interaction and thereby
determining the identity of the nucleotide.
[0010] The invention further provides: [0011] a method of
sequencing a target nucleic acid sequence, comprising: [0012] (a)
digesting an individual nucleotide from one end of the target
sequence using a processive exonuclease; [0013] (b) contacting the
nucleotide with a transmembrane protein pore so that the nucleotide
interacts with the pore; [0014] (c) measuring the current passing
through the pore during the interaction and thereby determining the
identity of the nucleotide; and [0015] (d) repeating steps (a) to
(c) at the same end of the nucleic acid sequence and thereby
determining the sequence of the nucleic acid; and [0016] a kit for
sequencing a nucleic acid, comprising: [0017] a cyclodextrin; and
[0018] a processive exonuclease.
[0019] The method of sequencing of the invention is a rapid and
simple DNA sequencing method at the single molecule level. It is
also a cheap method of sequencing DNA because it does not involve
the use of expensive reagents, such as fluorophores.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1 shows the .alpha.-hemolysin (M113R).sub.7 mutant and
heptakis-6-amino-.beta.-cyclodextrin (am.sub.7-.beta.CD).
A--sagittal cut through the .alpha.-hemolysin structure, position
113 is indicated by the arrow. B--spacefilled structure of
am.sub.7-.beta.CD. C--possible interaction of am.sub.7-.beta.CD
with .alpha.-hemolysin (M113R).sub.7
[0021] FIG. 2A shows dCMP detection. A--Current trace of single
(M113R).sub.7 mutant inserted in a phospholipid bilayer at +130 mV.
L1 identifies the current of the unoccupied protein nanopore. B--in
the presence of 40 .mu.M am.sub.7-.beta.CD in the trans chamber. L2
indicates the current level observed when am.sub.7-.beta.CD binds
temporarily inside the nanopore. C--dCMP 5 .mu.M is now added to
the cis chamber. L3 shows the current level that is observed when
dCMP binds to the temporary complex
(M113R).sub.7/am.sub.7-.beta.CD.
[0022] FIG. 2B shows the interaction of the .alpha.-hemolysin
(.alpha.HL) pore with
heptakis-(6-deoxy-6-amino)-.beta.-cyclodextrin (am.sub.7.beta.CD)
and dCMP. A--Model of the heptameric .alpha.HL pore (7AHL), in
which Met-113 has been substituted with Arg. A model of
am.sub.7.beta.CD in cross-section generated in ChemDraw Ultra has
been positioned manually at van der Waals distances from the Arg
side chains, which block the passage of the cyclodextrin when it
enters the pore from the trans side. When am.sub.7.beta.CD is
present inside the pore, two rings of positive charge, one ring of
seven primary amino groups contributed by the cyclodextrin, and a
second ring of seven arginine side-chains, are separated by
.about.10 .ANG.. Aminocyclodextrins have previously been shown to
bind nucleoside monophosphates with the phosphate group in an ionic
interaction with the protonated amino groups. It is possible that
the overall stability of such complexes is enhanced by p-cation
interactions between the nucleotide bases and the Arg side chains.
The dCMP molecule is positioned so that the phosphate group
interacts with the protonated amines of am.sub.7.beta.CD and the
cytosine ring interacts with the guanidinium groups of the Arg side
chains. B--Current trace from a single (M113R).sub.7 pore at +130
mV. L1 identifies the current flowing through the unoccupied
protein nanopore, which is shown as a model on the right.
C--Current trace after the addition of 40 .mu.M am.sub.7PCD to the
trans chamber. L2 indicates the current level observed when
am.sub.7.beta.CD is bound inside the nanopore. D--Current trace
after the addition of 5 .mu.M dCMP to the cis chamber. L3 shows the
current level that is observed when dCMP binds to the
(M113R).sub.7.cndot.am.sub.7.beta.CD complex.
[0023] FIG. 3 shows dXMP current amplitudes. Current trace of
single (M113R).sub.7 pore inserted in a phospholipid bilayer, at
+130 mV potential. 40 .mu.M am.sub.7-.beta.CD is present in the
trans chamber. A--dGMP 5 .mu.M is added to the cis chamber. The all
points histogram of the current trace is shown on the right
together with the structures of dGMP, dTMP (B), dAMP (C), and dCMP
(D).
[0024] FIG. 4 shows cyclodextrin current levels. Current trace of
single (M113R).sub.7 mutant inserted in a phospholipid bilayer with
40 .mu.M am.sub.7-.beta.CD present in the trans chamber at +130 mV.
L1 and L1' indicate two current levels of the unoccupied nanopore,
and L2 and L2' show two current levels resulting from the binding
of am.sub.7-.beta.CD to (M113R).sub.7. The insert shows the
amplitude histogram of the current trace with the peaks
corresponding to the current levels L1, L1'', L2, and L2'.
[0025] FIG. 5 shows single event analysis. A shows a single event
analysis histogram of the L3 current level from all four dXMP in
the same solution. B shows a single event analysis histogram of L3
originating from L2 only. 5 .mu.M of dGMP, dAMP, dCMP, and 10 .mu.M
of dTMP are present in the cis chamber.
[0026] FIG. 6 shows simultaneous detection of dXMP. A shows the
current trace of a single (M113R).sub.7 mutant inserted in a
phospholipid bilayer, +130 mV potential is applied between the
Ag/AgCl electrodes. The buffer is Tris-HCl 25 mM pH 8.0 with 1M
KCl. 40 .mu.M am.sub.7-.beta.CD is present in the trans chamber. 5
.mu.M of dGMP, dTMP, dAMP, and dCMP are added to the cis chamber.
The colored bands illustrate the amplitude distribution of each
dXMP. B displays an all point histogram from a current trace of
8000 binding events. Each peal is super-imposed with the
statistical distribution of each dXMP.
[0027] FIG. 7 shows the statistical method. Two Gaussian
distributions A and B overlap at the point of intersection I. The
area of Gaussian A beyond the point of intersection I is integrated
and represents the probability of population A to be identified as
population B.
DESCRIPTION OF THE SEQUENCE LISTING
[0028] SEQ ID NO: 1 shows the polynucleotide sequence that encodes
one subunit of .alpha.-hemolysin.
[0029] SEQ ID NO: 2 shows the amino acid sequence of one subunit of
.alpha.-hemolysin.
[0030] SEQ ID NO: 3 shows the polynucleotide sequence that encodes
one subunit of .alpha.-hemolysin M113H.
[0031] SEQ ID NO: 4 shows the amino acid sequence of one subunit of
.alpha.-hemolysin M113H.
[0032] SEQ ID NO: 5 shows the polynucleotide sequence that encodes
one subunit of .alpha.-hemolysin M113K.
[0033] SEQ ID NO: 6 shows the amino acid sequence of one subunit of
.alpha.-hemolysin M113K.
[0034] SEQ ID NO: 7 shows the polynucleotide sequence that encodes
one subunit of .alpha.-hemolysin M113R.
[0035] SEQ ID NO: 8 shows the amino acid sequence of one subunit of
.alpha.-hemolysin M113R.
[0036] SEQ ID NO: 9 shows the amino acid sequence of lambda
exonuclease. The sequence is one of three identical subunits that
assemble into a trimer.
DETAILED DESCRIPTION OF THE INVENTION
Method of Identifying an Individual Nucleotide
[0037] In a first embodiment, the present invention relates to a
method of identifying an individual nucleotide comprising
contacting the nucleotide with a transmembrane protein pore so that
the nucleotide interacts with the pore and measuring the current
passing through the pore during the interaction and thereby
determining the identity of the nucleotide. The invention therefore
involves stochastic sensing of an individual nucleotide. The
invention can be used to differentiate nucleotides of similar
structure on the basis of the different effects they have on the
current passing through a transmembrane protein pore. The invention
can also be used to determine whether or not a particular
nucleotide is present in a sample. The invention can also be used
to measure the concentration of a particular nucleotide in a
sample.
[0038] An individual nucleotide in accordance with the invention is
a single nucleotide. An individual nucleotide is one which is not
bound to another polynucleotide by a nucleotide bond. A nucleotide
bond involves one of the phosphate groups of a nucleotide being
bound to the sugar group of another nucleotide. An individual
nucleotide is typically one which is not bound by a nucleotide bond
to another polynucleotide sequence of at least 5, at least 10, at
least 20, at least 50, at least 100, at least 200, at least 500, at
least 1000 or at least 5000 nucleotides. For example, the
individual nucleotide has been digested from a target
polynucleotide sequence, such as a DNA or RNA strand. The
individual nucleotide may however be bonded or attached to other
chemical groups, such as fluorescent molecules or chemical groups
containing radioisotopes, e.g. .sup.125I, .sup.35S. The types of
nucleotides for identification in accordance with the invention are
discussed in more detail below.
[0039] The method may be carried out using any suitable
membrane/pore system in which a transmembrane protein pore is
inserted into a membrane. The method is typically carried out using
(i) an artificial membrane comprising a naturally-occurring or
recombinant transmembrane protein pore, (ii) an isolated,
naturally-occurring membrane comprising a recombinant transmembrane
protein pore, (iii) an isolated, naturally-occurring membrane
comprising a transmembrane protein pore or (iv) a cell expressing a
naturally-occurring or recombinant transmembrane protein pore. The
method is preferably carried out using an artificial membrane. The
membrane may comprise other transmembrane and/or intramembrane
proteins as well as other molecules in addition to the
transmembrane protein pore.
[0040] The method of the invention is typically carried out in
vitro.
Membrane
[0041] The membrane forms a barrier to the flow of ions and
nucleotides. The membrane is preferably a lipid bilayer. Lipid
bilayers suitable for use in accordance with invention can be made
using methods known in the art. For example, lipid bilayer
membranes can be formed using the method of Montal and Mueller
(1972). The method of the invention may be carried out using lipid
bilayers formed from any membrane lipid including, but not limited
to, phospholipids, glycolipids, cholesterol and mixtures thereof.
The lipid bilayer is preferably formed from
1,2-diphytanoyl-sn-glycero-3-phosphocholine.
[0042] Methods are known in the art for inserting pores into
membranes, such as lipid bilayers. For example, the pore may be
suspended in a purified form in a solution containing a lipid
bilayer such that it diffuses to the lipid bilayer and is inserted
by binding to the lipid bilayer and assembling into a functional
state. Alternatively, the pore may be directly inserted into the
membrane using the method described in M. A. Holden, H. Bayley. J.
Am. Chem. Soc. 2005, 127, 6502-6503.
Transmembrane Protein Pore
[0043] The method of the invention is carried out using a
transmembrane protein pore. A transmembrane protein pore is a
polypeptide that permits ions to flow from one side of the membrane
to the other along an electrochemical gradient. The pore preferably
permits the nucleotide to flow from one side of the membrane to the
other along an electrochemical gradient.
[0044] The pore is typically an oligomer. The pore is preferably
made up of several repeating subunits. The pore is preferably
pentameric or heptameric. The pore typically comprises a barrel or
channel through which the ions may flow.
[0045] The barrel or channel of the pore typically comprises amino
acids that facilitate interaction with the nucleotide. A pore for
use in accordance with the invention typically comprises one or
more positively charged amino acids, such as arginine, lysine or
histidine. These positively charged amino acids are preferably
located near the constriction of the barrel or channel. These amino
acids typically facilitate the interaction between the pore and the
nucleotide by interacting with the phosphate groups in the
nucleotide or by p-cation interaction with the base in the
nucleotide. The pore preferably has a ring of positively charged
amino acids, such as arginine, lysine or histidine, located near
the constriction of the barrel or channel. Each positively charged
amino acid is typically provided by each of the pore subunits.
[0046] Suitable pores for use in accordance with the invention
include, but are not limited to, .alpha.-hemolysin, porins and
leukocidins.
[0047] The preferred pore for use in the invention is
.alpha.-hemolysin or a variant thereof. The .alpha.-hemolysin pore
is formed of seven identical subunits (heptameric). The sequence of
one subunit of .alpha.-hemolysin is shown in SEQ ID NO: 2. A
variant is a heptameric pore in which one or more of the seven
subunits has an amino acid sequence which varies from that of SEQ
ID NO: 2 and which retains pore activity. 1, 2, 3, 4, 5, 6 or 7 of
the subunits in a variant .alpha.-hemolysin may have an amino acid
sequence that varies from that of SEQ ID NO: 2. The seven subunits
within a variant pore are typically identical but may be
different.
[0048] A preferred variant of .alpha.-hemolysin has one or more
positively charged amino acids, such as arginine, lysine or
histidine, located near the constriction of the barrel or channel.
The pore preferably has a ring of 4, 5, 6 or preferably 7
positively charged amino acids, such as arginine, lysine or
histidine, located near the constriction of the barrel or channel.
Each amino acid in the ring is typically provided by each of the
variant subunits. Variants typically include a positively charged
amino acid at position 113 of each subunit. The pore for use in the
invention is preferably .alpha.-hemolysin (M113K).sub.7 which
comprises seven subunits as shown in SEQ ID NO: 4 or preferably
.alpha.-hemolysin (M113H).sub.7 which comprises seven subunits as
shown in SEQ ID NO: 6 or most preferably .alpha.-hemolysin
(M113R).sub.7 which comprises seven subunits as shown in SEQ ID NO:
8.
[0049] The variant may be a naturally-occurring variant which is
expressed by an organism, for instance by a Staphylococcus
bacterium. Variants also include non-naturally occurring variants
produced by recombinant technology. Over the entire length of the
amino acid sequence of SEQ ID NO: 2, a subunit of a variant will
preferably be at least 50% homologous to that sequence based on
amino acid identity. More preferably, the subunit polypeptide may
be at least 55%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90% and more preferably
at least 95%, 97% or 99% homologous based on amino acid identity to
the amino acid sequence of SEQ ID NO: 2 over the entire sequence.
There may be at least 80%, for example at least 85%, 90% or 95%,
amino acid identity over a stretch of 200 or more, for example 230,
250, 270 or 280 or more, contiguous amino acids ("hard
homology").
[0050] Amino acid substitutions may be made to the amino acid
sequence of SEQ ID NO: 2, for example up to 1, 2, 3, 4, 5, 10, 20
or 30 substitutions. Conservative substitutions may be made, for
example, according to the following table. Amino acids in the same
block in the second column and preferably in the same line in the
third column may be substituted for each other:
TABLE-US-00001 NON-AROMATIC Non-polar G A P I L V Polar - uncharged
C S T M N Q Polar - charged D E H K R AROMATIC H F W Y
[0051] One or more amino acid residues of the amino acid sequence
of SEQ ID NO: 2 may alternatively or additionally be deleted. Up to
1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.
[0052] Variants may include subunits made of fragments of SEQ ID
NO: 2. Such fragments retain pore forming activity. Fragments may
be at least 50, 100, 200 or 250 amino acids in length. Such
fragments may be used to produce chimeric pores. A fragment
preferably comprises the pore forming domain of SEQ ID NO: 2.
[0053] Variants include chimeric protein pores comprising fragments
or portions of SEQ ID NO: 2. Chimeric protein pores are formed from
subunits each comprising fragments or portions of SEQ ID NO: 2. The
pore or channel part of a chimeric protein pore is typically formed
by the fragments or portions of SEQ ID NO: 2.
[0054] One or more amino acids may be alternatively or additionally
added to the polypeptides described above. An extension may be
provided at the N-terminus or C-terminus of the amino acid sequence
of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The
extension may be quite short, for example from 1 to 10 amino acids
in length. Alternatively, the extension may be longer, for example
up to 50 or 100 amino acids. A carrier protein may be fused to an
amino acid sequence according to the invention.
[0055] Standard methods in the art may be used to determine
homology. For example the UWGCG Package provides the BESTFIT
program which can be used to calculate homology, for example used
on its default settings (Devereux et al (1984) Nucleic Acids
Research 12, p387-395). The PILEUP and BLAST algorithms can be used
to calculate homology or line up sequences (such as identifying
equivalent residues or corresponding sequences (typically on their
default settings)), for example as described in Altschul S. F.
(1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol
Biol 215:403-10.
[0056] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighbourhood word score threshold (Altschul et al, supra).
These initial neighbourhood word hits act as seeds for initiating
searches to find HSP's containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10, M=5, N=4, and a comparison of both strands.
[0057] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two amino acid sequences would occur by
chance. For example, a sequence is considered similar to another
sequence if the smallest sum probability in comparison of the first
sequence to the second sequence is less than about 1, preferably
less than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0058] Pores used in accordance with the invention may be modified
for example by the addition of histidine residues to assist their
identification or purification or by the addition of a signal
sequence to promote their secretion from a cell where the
polypeptide does not naturally contain such a sequence. It may be
desirable to provide the polypeptides in a form suitable for
attachment to a solid support. For example, the pore may be
attached to a solid support in order to insert the pore into the
membrane.
[0059] A pore may be labelled with a revealing label. The revealing
label may be any suitable label which allows the pore to be
detected. Suitable labels include, but are not limited to,
fluorescent molecules, radioisotopes, e.g. .sup.125I, .sup.35S,
enzymes, antibodies, polynucleotides and linkers such as
biotin.
[0060] The pore may be isolated from a pore-producing organism,
such as Staphylococcus aureus, or made synthetically or by
recombinant means. For example, the pore may be synthesized by in
vitro translation transcription. The amino acid sequence of the
pore may be modified to include non-naturally occurring amino acids
or to increase the stability of the compound. When the pores are
produced by synthetic means, such amino acids may be introduced
during production. The pores may also be modified following either
synthetic or recombinant production.
[0061] The pores may also be produced using D-amino acids. In such
cases the amino acids will be linked in reverse sequence in the C
to N orientation. This is conventional in the art for producing
such proteins or peptides.
[0062] A number of side chain modifications are known in the art
and may be made to the side chains of the pores. Such modifications
include, for example, modifications of amino acids by reductive
alkylation by reaction with an aldehyde followed by reduction with
NaBH.sub.4, amidination with methylacetimidate or acylation with
acetic anhydride.
[0063] A recombinant transmembrane pore can be produced using
standard methods known in the art. Nucleic acid sequences encoding
a pore may be isolated and replicated using standard methods in the
art. Nucleic acid sequences encoding a pore may be expressed in a
bacterial host cell using standard techniques in the art. The pore
may be introduced into a cell by in situ expression of the
polypeptide from a recombinant expression vector. The expression
vector optionally carries an inducible promoter to control the
expression of the polypeptide.
[0064] Nucleic acid sequences encoding a pore may be isolated and
replicated using standard methods in the art. Chromosomal DNA may
be extracted from a pore-producing organism, such as Staphylococcus
aureus. The gene encoding the pore may be amplified using PCR
involving specific primers. The amplified sequence may then be
incorporated into a recombinant replicable vector such as a cloning
vector. The vector may be used to replicate the nucleic acid in a
compatible host cell. Thus nucleic acid sequences encoding a pore
may be made by introducing a polynucleotide encoding a pore into a
replicable vector, introducing the vector into a compatible host
cell, and growing the host cell under conditions which bring about
replication of the vector. The vector may be recovered from the
host cell. Suitable host cells for cloning of polynucleotides
encoding a pore are known in the art and described in more detail
below.
[0065] The nucleic acid sequence encoding a pore may be cloned into
suitable expression vector. In an expression vector, the nucleic
acid sequence encoding a pore is typically operably linked to a
control sequence which is capable of providing for the expression
of the coding sequence by the host cell. Such expression vectors
can be used to express a pore.
[0066] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. Multiple copies of the same
or different pore genes may be introduced into the vector.
[0067] The expression vector may then be introduced into a suitable
host cell. Thus the method of the invention may be carried out on a
cell produced by introducing a nucleic acid sequence encoding a
pore into an expression vector, introducing the vector into a
compatible bacterial host cell, and growing the host cell under
conditions which bring about expression of the nucleic acid
sequence encoding the pore. Alternatively, the recombinant pore
produced in this manner may be isolated from the bacterial host
cell and inserted into another membrane.
[0068] The vectors may be for example, plasmid, virus or phage
vectors provided with an origin of replication, optionally a
promoter for the expression of the said nucleic acid sequence and
optionally a regulator of the promoter. The vectors may contain one
or more selectable marker genes, for example a tetracycline
resistance gene. Promoters and other expression regulation signals
may be selected to be compatible with the host cell for which the
expression vector is designed. A T7, trc, lac, ara or .lamda..sub.L
promoter is typically used.
[0069] The host cell typically expresses the pore at a high level.
Host cells transformed with a nucleic acid sequence encoding a pore
will be chosen to be compatible with the expression vector used to
transform the cell. The host cell is typically bacterial and
preferably Escherichia coli. Any cell with a .lamda. DE3 lysogen,
for example C41 (DE3), BL21 (DE3), JM109 (DE3), B834 (DE3), TUNER,
Origami and Origami B, can express a vector comprising the T7
promoter.
[0070] A pore may be produced in large scale following purification
by any protein liquid chromatography system from pore-producing
organisms or after recombinant expression as described above.
Typical protein liquid chromatography systems include FPLC, AKTA
systems, the Bio-Cad system, the Bio-Rad BioLogic system and the
Gilson HPLC system. The naturally-occurring or
recombinantly-produced pore may then be inserted into a
naturally-occurring or artificial membrane for use in accordance
with the invention.
[0071] The method of the invention may employ any one of the pores
described above.
Interaction Between the Pore and Nucleotide
[0072] The nucleotide may be contacted with the pore on either side
of the membrane. The nucleotide may be introduced to the pore on
either side of the membrane. The nucleotide is preferably contacted
with the pore on a side of the membrane that allows ions to enter
the pore and flow across the membrane along an electrochemical
gradient. The nucleotide is preferably contacted with a side of the
membrane that allows the nucleotide to pass through the pore to the
other side of the membrane. For example, the nucleotide is
contacted with an end of the pore which in its native environment
allows the entry of ions or small molecules, such as nucleotides,
into the barrel or channel of the pore such that the ions or small
molecules may pass through the pore.
[0073] The nucleotide may interact with the pore in any manner and
at any site. The nucleotide preferably reversibly binds to the
pore. The nucleotide more preferably reversibly binds to the barrel
or the channel of the pore. The nucleotide most preferably
reversibly binds to the channel or barrel of the pore as it passes
through the pore across the membrane.
[0074] During the interaction between the nucleotide and the pore,
the nucleotide affects the current flowing through the pore in a
manner specific for that nucleotide. For example, a particular
nucleotide will reduce the current flowing through the pore for a
particular time period and to a particular extent. Control
experiments may be carried out to determine the effect a particular
nucleotide has on the current flowing through the pore. Results
from carrying out the method of the invention on a test sample can
then be compared with those derived from such a control experiment
in order to identify a particular nucleotide in the sample or
determine whether a particular nucleotide is present in the sample.
The frequency at which the current flowing through the pore is
affected in a manner indicative of a particular nucleotide can be
used to determine the concentration of that nucleotide in the
sample.
Apparatus
[0075] The method may be carried out using any apparatus that is
suitable for investigating a membrane/pore system in which a
transmembrane protein pore is inserted into a membrane. The method
may be carried out using any apparatus that is suitable for
stochastic sensing. For example, the apparatus comprises a chamber
comprising an aqueous solution and a barrier that separates the
chamber into two sections. The barrier has an aperture in which the
membrane comprising the pore is formed. The nucleotide may be
contacted with the pore by introducing the nucleotide into the
chamber. The nucleotide may be introduced into either of the two
sections of the chamber.
[0076] The method of the invention involves measuring the current
passing through the pore during interaction with the nucleotide.
Therefore the apparatus also comprises an electrical circuit
capable of applying and measuring an electrical signal across the
membrane and pore. The method may be carried out using a patch
clamp or a voltage clamp. The method preferably involves the use of
a patch clamp. The Example discloses one way of carry out a patch
clamp method.
Molecular Adaptor
[0077] The transmembrane pore preferably comprises a molecular
adaptor that facilitates the interaction between the pore and the
nucleotide. The adaptor typically has an effect on the physical or
chemical properties of the pore that improves its interaction with
the nucleotide. The adaptor typically alters the charge of the
barrel or channel of the pore or specifically interacts with or
binds to the nucleotide thereby facilitating its interaction with
the pore. The adaptor preferably interacts with one or more
phosphate groups on the nucleotide or interacts with the base in
the nucleotide by p-cation interaction. The adaptor may mediate the
interaction between the nucleotide and the pore. For instance, the
nucleotide may reversibly bind to the pore via the adaptor.
Alternatively, the adaptor may interact with the nucleotide in
conjunction with the pore. For instance, the nucleotide may
reversibly bind to both the pore and the adaptor. The adaptor
preferably constricts the barrel or channel so that it may interact
with the nucleotide.
[0078] The adaptor itself may reversibly interact with the pore and
may therefore move in and out of the barrel or channel of the pore.
Alternatively, the adaptor may be covalently attached to the barrel
or channel of the pore so that it cannot leave.
[0079] The adaptor typically has a ring of amino groups. The
adaptor preferably has a ring of seven amino groups. This ring of
amino groups may interact with the nucleotide in combination with a
ring of positively charged amino acids in the constriction of the
barrel or channel of the pore.
[0080] One suitable adaptor is cyclodextrin. The adaptor is
preferably heptakis-6-amino-.beta.-cyclodextrin
(am.sub.7-.beta.-CD).
Nucleotide
[0081] The method of the invention may be used to identify any
nucleotide. The nucleotide can be naturally-occurring or
artificial. A nucleotide typically contains a nucleobase, a sugar
and at least one phosphate group. The nucleobase is typically
heterocyclic. Suitable nucleobases include purines and pyrimidines
and more specifically adenine, guanine, thymine, uracil and
cytosine. The sugar is typically a pentose sugar. Suitable sugars
include, but are not limited to, ribose and deoxyribose. The
nucleotide is typically a ribonucleotide or deoxyribonucleotide.
The nucleotide typically contains a monophosphate, diphosphate or
triphosphate.
[0082] Suitable nucleotides include, but are not limited to,
adenosine monophosphate (AMP), adenosine diphosphate (ADP),
adenosine triphosphate (ATP), guanosine monophosphate (GMP),
guanosine diphosphate (GDP), guanosine triphosphate (GTP),
thymidine monophosphate (TMP), thymidine diphosphate (TDP),
thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine
diphosphate (UDP), uridine triphosphate (UTP), cytidine
monophosphate (CMP), cytidine diphosphate (CDP), cytidine
triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic
guanosine monosphosphate (cGMP), deoxyadenosine monophosphate
(dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine
triphosphate (dATP), deoxyguanosine monophosphate (dGMP),
deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate
(dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine
diphosphate (dTDP), deoxythymidine triphosphate (dTTP),
deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),
deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate
(dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine
triphosphate (dCTP). The nucleotide is preferably AMP, TMP, GMP,
UMP, dAMP, dTMP, dGMP or dCMP.
[0083] The nucleotide may be derived from the digestion of a
nucleic acid sequence such as ribonucleic acid (RNA) or
deoxyribonucleic acid. Individual nucleotides from a single nucleic
acid sequence may be contacted with the pore in a sequential manner
in order to sequence the whole or part of the nucleic acid.
Sequencing nucleic acids in accordance with the second embodiment
of the invention is discussed in more detail below.
[0084] The nucleotide is typically unmodified, such as when the
nucleotide is derived from the digestion of a nucleic acid
sequence. Alternatively, the nucleotide may be modified or damaged.
The nucleotide is typically methylated. The nucleotide may be
labelled with a revealing label. The revealing label may be any
suitable label which allows the nucleotide to be detected. Suitable
labels include fluorescent molecules, radioisotopes, e.g.
.sup.125I, .sup.35S, and linkers such as biotin.
[0085] The nucleotide is typically present in any suitable
biological sample. The invention is typically carried out on a
sample that is known to contain or suspected of containing one or
more nucleotides. The invention may be carried out on a sample that
contains one or more nucleotides whose identity is unknown.
Alternatively, the invention may be carried out on a sample to
confirm the identity of one or more nucleotides whose presence in
the sample is known or expected. The invention may be carried out
in vitro on a sample obtained from or extracted from any organism
or microorganism. The organism or microorganism is typically
prokaryotic or eukaryotic and typically belongs to one the five
kingdoms: plantae, animalia, fungi, monera and protista. The
invention may be carried out in vitro on a sample obtained from or
extracted from any virus. The sample is preferably a fluid sample.
The sample typically comprises a body fluid of the patient. The
sample may be urine, lymph, saliva, mucus or amniotic fluid but is
preferably blood, plasma or serum. Typically, the sample is human
in origin, but alternatively it may be from another mammal animal
such as from commercially farmed animals such as horses, cattle,
sheep or pigs or may alternatively be pets such as cats or
dogs.
[0086] The sample is typically processed prior to being assayed,
for example by centrifugation or by passage through a membrane that
filters out unwanted molecules or cells, such as red blood cells.
The sample may be measured immediately upon being taken. The sample
may also be typically stored prior to assay, preferably below
-70.degree. C.
Conditions
[0087] The method of the invention involves the measuring of a
current passing through the pore during interaction with the
nucleotide. Suitable conditions for measuring ionic currents
through transmembrane protein pores are known in the art and
disclosed in the Example. The method is carried out with a voltage
applied across the membrane and pore. The voltage used is typically
from +50 mV to +200 mV. The voltage used is preferably from +70 mV
to +150 mV, from +85 mV to +145 mV or from +100 mV to +140 mV. The
voltage used is preferably about +130 mV for deoxy-ribo nucleotides
5' monophosphate, such as dAMP, dTMP, dGMP and dCMP, and +110 mV
for ribo nucleotides 5' monophosphate, such as AMP, TMP, GMP and
UMP.
[0088] The method is carried out in the presence of any alkali
metal chloride salt. In the exemplary apparatus discussed above,
the salt is present in the aqueous solution in the chamber.
Potassium chloride (KCl), sodium chloride (NaCl) or caesium
chloride (CsCl) is typically used. KCl is preferred. The salt
concentration is typically from 0.1 to 2M, from 0.3 to 1.9M, from
0.5 to 1.8M, from 0.7 to 1.7M, from 0.9 to 1.6M or from 1M to 1.4M.
The salt concentration is preferably about 1M.
[0089] The method is typically carried out in the presence of a
buffer. In the exemplary apparatus discussed above, the buffer is
present in the aqueous solution in the chamber. Any buffer may be
used in the method of the invention. One suitable buffer is
Tris-HCl buffer. The method is typically carried out at a pH of
from 7.5 to 12.0, from 7.6 to 11.0, from 7.7 to 10.0, from 7.8 to
9.5, from 8.0 to 9.0 or from 8.0 to 8.5. The pH used is preferably
about 8.0.
[0090] The method is typically carried out at from 14.degree. C. to
100.degree. C., from 15.degree. C. to 90.degree. C., from
16.degree. C. to 80.degree. C., from 17.degree. C. to 70.degree.
C., from 18.degree. C. to 60.degree. C., 19.degree. C. to
50.degree. C., or from 20.degree. C. to 40.degree. C. The method is
preferably carried out at room temperature.
[0091] The method is preferably carried out at +130 mV at pH 8.0,
1M KCl for deoxy-ribo nucleotides 5' monophosphate, such as dAMP,
dTMP, dGMP and dCMP, and at +110 mV at pH 8.0, 1M KCl for ribo
nucleotides 5' monophosphate, such as AMP, TMP, GMP and UMP.
Method of Sequencing Nucleic Acids
[0092] In a second embodiment, the invention relates to a method of
sequencing a target nucleic acid sequence, comprising (a) digesting
an individual nucleotide from one end of the target sequence using
a processive exonuclease; (b) contacting the nucleotide with a
transmembrane protein pore so that the nucleotide interacts with
the pore; (c) measuring the current passing through the pore during
the interaction and thereby determining the identity of the
nucleotide; and (d) repeating steps (a) to (c) at the same end of
the nucleic acid sequence and thereby determining the sequence of
the nucleic acid. Hence, the second embodiment involves stochastic
sensing of each single nucleotide of a nucleic acid sequence in a
successive manner in order to sequence the nucleic acid. The whole
or only part of the nucleic acid may be sequenced using the method
of the second embodiment. The nucleic acid can be
naturally-occurring or artificial. For instance, the method of the
second embodiment may be used to verify the sequence of a
manufactured oligonucleotide. The method of the second embodiment
is typically carried out in vitro.
[0093] Steps (b) and (c) of the method of the second embodiment are
generally identical to the steps carried out in the method of the
first embodiment discussed above. All of the discussion above
concerning the first embodiment, and in particular concerning the
membranes, apparatus, pores, molecular adaptors, nucleotides and
conditions that may be used in the first embodiment, equally
applies to the second embodiment. The nucleic acid in the second
embodiment is typically present in any biological sample as
discussed above for the first embodiment. The method of the second
embodiment may be carried out on a sample which contains one or
more nucleic acids whose sequence is unknown. Alternatively the
method of the second embodiment may be carried out on a sample to
confirm the identity of nucleic acids whose presence in the sample
is known or is expected. The nucleic acid sequence is typically
amplified prior to being sequenced using the method of the second
embodiment.
Processive Exonuclease
[0094] The method of the second embodiment involves contacting the
nucleic acid sequence with a processive exonuclease to release
individual nucleotides from one end of the nucleic acid. Processive
exonucleases are enzymes that typically latch onto one end of a
nucleic acid sequence and digest the sequence one nucleotide at a
time from that end. The processive exonuclease can digest the
nucleic acid in the 5' to 3' direction or 3' to 5' direction. The
end of the nucleic acid to which the processive exonuclease binds
is typically determined through the choice of enzyme used and/or
using methods known in the art. Hydroxyl groups or cap structures
at either end of the nucleic acid sequence may typically be used to
prevent or facilitate the binding of the processive exonuclease to
a particular end of the nucleic acid sequence.
[0095] Any processive exonuclease enzyme may be used in the method
of the invention. The preferred enzyme for use in the method of the
invention is lambda exonuclease. The sequence of one subunit of
lambda exonuclease is shown in SEQ ID NO: 9. Three identical
subunits interact to form a trimer exonuclease. Variants of lambda
exonuclease are enzymes formed of polypeptide subunits which have
an amino acid sequence which varies from that of SEQ ID NO: 9 and
which retain processive exonuclease activity. The variants may vary
from SEQ ID NO: 9 in the same manner and to the same extent as
discussed for variants of SEQ ID NO: 2 above. A variant preferably
comprises the domains responsible for binding to the nucleic acid
and for digesting the nucleic acid (catalytic domain). A variant
preferably has a reduced rate of enzyme activity and/or higher salt
tolerance compared to the wild-type enzyme. The processive
exonuclease may be produced using any of the methods discussed
above for the production of transmembrane protein pores.
[0096] The method of the second embodiment involves contacting the
nucleic acid sequence with the processive exonuclease so that the
nucleotides are digested from the end of the nucleic acid at a rate
that allows identification of each individual nucleotide in
accordance with the first embodiment of the invention. Methods for
doing this are well known in the art. For example, Edman
degradation is used to successively digest single amino acids from
the end of polypeptide such that they may be identified using High
Performance Liquid Chromatography (HPLC). A homologous method may
be used in the present invention.
[0097] The processive exonuclease is preferably covalently attached
to the transmembrane protein pore. Methods for covalently attaching
the processive exonuclease to the pore are well known in the
art.
[0098] The rate at which the processive exonuclease must function
in the method of the second embodiment is typically slower than the
optimal rate of a wild-type processive exonuclease. A suitable rate
of activity of the processive exonuclease in the method of the
second embodiment involves digestion of from 0.5 to 1000
nucleotides per second, from 0.6 to 500 nucleotides per second, 0.7
to 200 nucleotides per second, from 0.8 to 100 nucleotides per
second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10
nucleotides per second. The rate is preferably 1, 10, 100, 500 or
1000 nucleotides per second. A suitable rate of processive
exonuclease activity can be achieved in various ways. For example,
variant processive exonucleases with a reduced optimal rate of
activity may be used in accordance with the invention.
[0099] The activity of processive exonucleases is typically pH
dependent such that their activity falls as pH is reduced. Hence,
the method of the second embodiment is typically carried out at a
pH of from 7.5 to 8.0 or from 7.7 to 8.0. The pH used is preferably
about 8.0.
[0100] The rate of activity of processive exonucleases typically
falls as salt concentration rises. However, very high salt
concentrations typically have a detrimental effect on the activity
of the enzyme. Another way of limiting the rate of the enzyme is to
carry out the method of the second embodiment at a salt
concentration that reduces the rate of the activity of the enzyme
without adversely affecting its activity. For example, the method
of the second embodiment may be carried out at a salt concentration
of from 0.5 to 1M. The salt concentration is preferably about
1M.
Kits
[0101] In a third embodiment, the invention also relates to kits
that may be used to carry out the second embodiment of the
invention. The kits are therefore suitable for sequencing nucleic
acids. The kits comprises a cyclodextrin and a processive
exonuclease. The cyclodextrin is preferably
heptakis-6-amino-.beta.-cyclodextrin. The processive exonuclease
may be any of those discussed above with reference to the second
embodiment. The kit preferably further comprises a transmembrane
protein pore. The pore may be any of those discussed above with
reference to the first embodiment.
[0102] The kit may additionally comprise one or more other reagents
or instruments which enable any of the embodiments of the method
mentioned above to be carried out. Such reagents or instruments
include one or more of the following: suitable buffer(s) (aqueous
solutions), means to obtain a sample from a subject (such as a
vessel or an instrument comprising a needle), means to amplify
nucleic acid sequences, a membrane as defined above or voltage or
patch clamp apparatus. Reagents may be present in the kit in a dry
state such that a fluid sample resuspends the reagents. The kit may
also, optionally, comprise instructions to enable the kit to be
used in the method of the invention or details regarding which
patients the method may be used for. The kit may, optionally,
comprise nucleotides.
[0103] The following Example illustrates the invention:
EXAMPLE
[0104] In order to bring the size of the ionic conducting path of
the .alpha.-hemolysin (M113R).sub.7 mutant (FIG. 1A) closer to the
size of the nucleotide to be detected, the diameter of the nanopore
was reduced by fitting a cyclodextrin near the constriction of the
pore. The heptakis-6-amino-.beta.-cyclodextrin (am.sub.7-.beta.CD)
(FIG. 1B), which has seven amino groups in the primary positions,
was used. When the cyclodextrin is inside the pore (FIG. 1C), in
conjunction with the seven arginines in position 113 on the protein
mutant, one ring of seven amino groups on one side, and a second
ring of seven arginine groups are present within a short distance
from each other in the narrowest area of the passage through the
pore. This amino/arginine ring structure has the property of
binding phosphate groups reversibly thus immobilising the XMP and
dXMP in the pore for 5 to 30 ms. These binding events are clearly
detectable through the resulting change in current amplitude.
1. Material and Methods
[0105] .alpha.-hemolysin mutant (M113R).sub.7 was expressed and
purified as previously described (Cheley, S., L.-Q. Gu, et al.
(2002), Chem. & Biol. 9: 829-838).
Chemicals
[0106] 1,2-diphytanoyl-sn-glycero-3-phosphocholine from Avanti
Polar Lipids Inc. Pentane was purchased from JT Baker, and
hexadecane 99+% from Sigma-Aldrich.
Heptakis(6-deoxy-6-amino)-.beta.-cyclodextrin.cndot.HCl >99% was
purchased from CYCLOLAB Ltd Budapest, Hungary. 2-deoxy-guanosine 5'
monophosphate sodium salt 99% was purchased from Acros,
2-deoxy-cytosine 5' monophosphate di-sodium salt >95%,
2-deoxy-thymidine 5' monophosphate di-sodium salt >97%, and
2-deoxy-adenosine 5 monophosphate di-sodium salt >95% from
Fluka. Uridine 5' monophosphate di-sodium salt 99%, and cytosine 5'
monophosphate acid >98% were bought from Fluka. Adenosine 5'
monophosphate acid 99%, and guanosine 5' monophosphate di-sodium
salt 97% were purchased from Acros. Trizma Base 99.9% was purchased
from Sigma-Aldrich, and concentrated HCl analytical reagent grade
from Fisher Scientific. Potassium chloride 99%, and sodium chloride
99.9% were purchased from Sigma-Aldrich. Potassium bromide 99.5%
and cesium chloride 99% were purchased from Fluka.
Equipment
[0107] A patch clamp amplifier Axopatch 200B from Axon instruments
was used with a computer equipped with a Digidata 1200 A/D
converter (Axon instruments). A Teflon chamber was used. Data was
collected in pClamp 9.2, and analyzed in Clampfit 9.0. Plots and
graphs were obtained with Microcal Origin 6.0, and integration were
run on a personal calculator.
Experimental Conditions
[0108] Lipid bilayer membranes were formed from
1,2-diphytanoyl-sn-glycero-3-phosphocholine by the method of Montal
and Mueller (1972), on 100-150 .mu.m diameter orifice in a 20 .mu.m
polycarbonate film (20 .mu.m thickness from Goodfellow, Malvern
Pa.) separating the trans and the cis chamber. The cis side of the
chamber was at ground, and the trans side of the chamber was
connected to the head stage. The potential refers to the potential
value of the trans side electrode. The adaptor molecule was added
to the trans side, the .alpha.-hemolysin mutant and the analyte
molecules were added to the cis side. dXMP experiments were carried
out at +130 mV, XMP experiments at +110 mV. All experiments
reported here were obtained at pH 8.0 Tris-HCl 25 mM in 1 M KCl.
Fresh aliquots of nucleotide solution were used everyday.
Experiments were carried out at room temperature 22.5+/-2.degree.
C. unless stated otherwise.
2. Results
2-Deoxy-Nucleotide 5' Monophosphates Partially Block Homoheptameric
Pores Formed by (M113R).sub.7/Heptakis 6 Amino
.beta.-Cyclodextrin
[0109] Single-channel recordings were carried out on the
homo-heptameric pores formed from .alpha.HL-M113R with
am.sub.7-.beta.CD applied from the trans side (FIGS. 2A and 2B). In
the absence of am.sub.7-.beta.CD, the pore remained permanently
open (FIGS. 2A and 2B, B) with a unitary current (L1) of 145.+-.5
pA (+130 mV) in 1M KCl in pH 8.0 Tris-HCl 25 mM buffer. The
addition of 40 .mu.M am.sub.7-.beta.CD in the trans chamber alone
leads to reversible blocking events to a current level of 65.+-.5
pA (L2 in FIG. 2A, B and FIG. 2B, C). Upon addition of 5 .mu.M dCMP
to the cis chamber, a third current level is observed at 22.+-.1 pA
(L3 in FIG. 2A, C and FIG. 2B, D) originating from current level
L2. L3 represents the binding of dCMP to the complex of
(M113R).sub.7 with am.sub.7-.beta.CD. Addition of dXMP at up to 300
.mu.M to the trans instead of the cis side of the chamber did not
lead to any alteration of the cyclodextrin binding conductance
states (not shown).
[0110] In the experimental conditions described above, current
blocking events due to cyclodextrin binding were observed when
unmodified .beta.-cyclodextrin was added to the trans chamber (40
.mu.M) in the presence of a .alpha.HL-M113R single nanopore.
However, no further current blocking events were observed, when
dXMPs (up to 300 .mu.M) were added either to the trans or the cis
chamber (not shown).
[0111] In the absence of am.sub.7-.beta.CD in the trans chamber,
blocking events (<1 ms) were observed when minimum
concentrations of 300 .mu.M dGMP or dTMP were added to the cis
chamber (not shown). These events are not observed over the
timescale of the experiment at 5 .mu.M dXMP or XMP.
[0112] Adding am.sub.7-.beta.CD in the trans chamber while
measuring the current through a wild type .alpha.-HL single channel
led to cyclodextrin binding events, but no further alterations of
the current were observed when adding dXMP to either the cis or the
trans chamber.
2-Deoxy-Nucleotide 5' Monophosphate can be Identified from the
Amplitude of the Partial Block of Homoheptameric Pores Formed by
(M113R).sub.7/Heptakis 6 Amino .beta.-Cyclodextrin
[0113] The partial block of the transient complex
(M113R).sub.7/am.sub.7-.beta.CD differed in amplitude depending on
which dXMP was added to the cis chamber (FIG. 3). Addition of dGMP
(5 .mu.M) to the cis side displayed an average blocking to a
current level of 16 pA (FIG. 3A). The all points amplitude
histogram of the trace shown in FIG. 3A is shown to the right of
the trace together with the structure of dGMP. The other
nucleotides all display different amplitudes as shown in FIG. 3B
for dTMP, 3C for dAMP, and 3D for dCMP. Out of the four dXMP, dGMP
blocks the most current.
[0114] The current amplitudes from independent experiments
displayed some variations that originated from individualities of
the protein nanopore. The average current, at +130 mV, for a
(M113R).sub.7 is 139 pA, but some channels display currents as high
as 147 pA and as low as 131 pA. Therefore, to compare current
traces current traces were normalized from different experiments
between 0 current and the (M113R).sub.7/am.sub.7-.beta.CD current
level set to 65 pA.
[0115] The dwell time (.tau..sub.off) of each dXMP was calculated
over 500 events from each of 3 independent experiments (Table
1).
TABLE-US-00002 TABLE 1 Dwell time in ms of dGMP, dTMP, dAMP, and
dCMP averaged from three independent measurements. dGMP dTMP dAMP
dCMP .tau..sub.off(ms) 9.8 .+-. 0.2 19.8 .+-. 0.8 7.1 .+-. 0.2 10.5
.+-. 0.4
Cyclodextrin Current Levels
[0116] At pH 8.0, the mutant (M113R).sub.7 exhibits two current
levels L1/L1' when the protein channel is unoccupied (FIG. 4). The
cyclodextrin adapter can bind to the protein regardless of which
current level L1/L1' the protein is in.
[0117] Two current levels are observed when recording the current
level of the (M113R).sub.7 nanopore (FIG. 4). L1 is the main
current level, as shown in the insert of FIG. 4. The binding of
am.sub.7-.beta.CD to the nanopore leads to two current levels
represented by L2 and L2' (three levels are observed at pH 7.5, not
shown). The binding of am.sub.7-.beta.CD to the protein nanopore
occurs independently of current level L1 or L1'. L2 is the main
conductance level observed when am.sub.7-.beta.CD is bound to
(M113R).sub.7 and it originates from both L1 and L1' conductance
levels of the empty nanopore with no apparent correlation. The
current level L2' observed when am.sub.7-.beta.CD is bound to
(M113R).sub.7 accounts for less than 15% of the conductance when
the cyclodextrin adapter is bound (see insert FIG. 4).
[0118] The nucleotide binding events sometimes vary in amplitude as
a result of which current level L2 and L2' they originate from. A
0.5 pA shift was observed depending on which of L2 or L2' the dXMP
binding event originates from (not shown). It leads to an increased
overlap of the nucleotide binding event histogram (FIG. 5).
[0119] It is possible to analyze manually each recording in order
to remove each analyte binding event stemming from the bound
cyclodextrin current level L2' described in FIG. 3. FIG. 5 shows
the difference between the single event analysis histogram obtained
from an unmodified dXMP detection current recording (FIG. 5A), and
the same recording where analyte binding events stemming off level
L2' (FIG. 4) have been removed. The two histograms display the same
four peaks corresponding to dGMP, dTMP, dAMP, and dCMP. The
amplitude of the peaks in FIG. 5A is larger than in 5B because the
analyte binding events stemming off L2' have been removed,
therefore the histogram originates from fewer events. The
separation between each peak seems better in 5B than in 5A.
However, removing these events from the recording did not yield a
complete separation of each peak (FIG. 5B). As a result, the
cyclodextrin current levels L2 and L2' shown in FIG. 4 were not
taken into account in the single event analysis histograms, and
resulting statistics reported hereafter.
2-Deoxy Nucleotide 5' Monophosphates can be Identified from the
Amplitude of the Partial Block of Homoheptameric Pores Formed by
the Transient Complex (M113R).sub.7/Heptakis 6 Amino
.beta.-Cyclodextrin
[0120] The partial block of (M113R).sub.7/am.sub.7-.beta.CD pores
proved to differ in amplitude depending on which dXMP was added to
the cis chamber. The different amplitudes could be resolved when
dGMP, dTMP, dAMP, and dCMP were added to the cis chamber
simultaneously (FIG. 5).
[0121] FIG. 6A shows the current amplitude for a mixed solution of
all 4 nucleotides from a single experiment. Colored bands are
superimposed onto the recorded current trace in order to illustrate
the amplitude distribution of each dXMP. FIG. 6B shows an amplitude
histogram of a current trace of 8000 events assumed to be the
amplitude distribution generated by each nucleotide. The amplitude
histogram is superimposed with Gaussian distributions. The fit is
obtained from the peak current value given by this experiment as
the distribution mean value and the .sigma. value that was obtained
from fitting and averaging the distribution of each nucleotide
independently (Table 2). From fitting the current traces with
Gaussian distributions, the probabilities of identification for
each nucleotide was established.
Statistical Methods
[0122] Current traces of (M113R).sub.7 in the presence of
am.sub.7-.beta.CD on the trans side and one of the analyte
nucleotides on the cis side were digitally filtered at 300 Hz (low
pass Gaussian filter), and an all points amplitude histogram was
constructed. These histograms display a large peak corresponding to
the current amplitude that is observed when the am.sub.7-.beta.CD
binds to the (M113R).sub.7 .alpha.-hemolysin mutant (corresponding
to L2 in FIG. 1). This current amplitude varies between protein
channels within 5% from one experiment to another. For this reason,
the all points amplitude histograms were normalised between 0
current, and the main cyclodextrin peak set at 65 pA. In the
normalised histogram, the nucleotide peak was fitted to a Gaussian
distribution. The mean and sigma (.sigma.) values of the same
nucleotide were averaged from at least 3 independent experiments
each containing 1000 events (values listed in Table 2).
TABLE-US-00003 TABLE 2 Average values of the distributions of each
nucleotide from three independent experiments all normalised
between 0 and 65 pA. Average G 16.0, .sigma. = 0.64 T 17.4, .sigma.
= 0.41 A 18.4, .sigma. = 0.54 C 20.0, .sigma. = 0.51
[0123] The probabilities for the reading of each base were
determined from experiments with all 4 nucleotides present
simultaneously over at least 3000 nucleotide binding events in each
trace. The traces were filtered (300 Hz low pass Gaussian digital
filter) and normalised between 0 and the cyclodextrin peak at 65 pA
as described above for the individual nucleotide experiments. The
peak values of each nucleotide were averaged from 5 independent
experiments (Table 3).
TABLE-US-00004 TABLE 3 Peak values of each nucleotide where all 4
nucleotides are present from 5 independent experiments. The last
column displays the average value of each peak for which the
overlap of the Gaussian distributions are integrated. Average G(pA)
16.2 .+-. 0.5 T(pA) 17.6 .+-. 0.6 A(pA) 18.6 .+-. 0.6 C(pA) 20.2
.+-. 0.5
[0124] In experiments where all 4 nucleotides are present, the
Gaussians from all 4 nucleotides have an overlap. The statistics
were calculated for the binding signal from one nucleotide to be
identified as itself or another nucleotide from the level of
overlap between the Gaussian distribution of this nucleotide and
that of its neighbors.
[0125] The point of intersection between the two Gaussians is
calculated form the respective peak positions (given by the
experiment with mixed nucleotides), and .sigma. values for each
distribution (given by the fitting of individual nucleotide
experiments). The accuracy probability is given by integrating the
area of the Gaussian that is beyond the intercept value with the
neighboring Gaussian (FIG. 6, Table 4). The first column of Table 4
is the nucleotide that interacts with the nanopore, and the first
row is what is read from the corresponding current amplitude.
TABLE-US-00005 TABLE 4 Shows the probability of the added
nucleotide (vertical) to be detected as itself or another
nucleotide (horizontal). G.sub.read T.sub.read A.sub.read
C.sub.read G.sub.added 0.88 0.12 0 0 T.sub.added 0.06 0.83 0.11 0
A.sub.added 0 0.19 0.74 0.07 C.sub.added 0 0 0.06 0.94
3. Conclusion
[0126] The results presented indicate that stochastic sensing is a
promising alternative for the identification of single nucleotides.
It also means that exonuclease sequencing can be used as a cheap,
rapid, and simple DNA sequencing method at the single molecule
level. Exonucleas sequencing is also a cheap method of sequencing
DNA because it does not require expensive reagents, such as
fluorophores.
[0127] All points histograms are a sufficient analysis method to
identify each nucleotide with an accuracy ranging from 74 to 94%
(Table 4). The dwell time values of the XMP and dXMP are too
similar in the conditions to further differenciate each analyte.
The statistics drawn from the amplitude histograms can be further
improved by compensating for the cyclodextrin current levels as
shown in FIG. 4. The current amplitude difference between each dXMP
is about 1 pA. This resolution depends on a number of parameters as
follows.
Voltage Dependence
[0128] The binding events are voltage dependent. At 50 mV, very few
binding events are observed, suggesting that a minimum field is
required to drive the dXMP and XMP to the binding site. At +150 and
+200 mV the amplitudes no longer allow to differentiate the
nucleotides. +130 mV proved to be the best voltage for deoxy-ribo
nucleotides 5' monophosphate, and +110 mV yielded the best
resolution for ribo nucleotides 5' monophosphate.
Salt Concentration
[0129] Tris-HCl pH 8 buffer 0.5, 1, and 2M KCl were tested. From
the all points amplitude histograms, the best resolution between
the peaks was obtained at 1M KCl.
pH Dependence
[0130] The current amplitudes are pH dependent, Tris-HCl buffer 1M
KCl at pH 7.5, 8.0, 8.2, 8.5, 9.0, and 9.5 were tested. At pH 8.0
and above two current levels are observed upon binding of
am.sub.7-.beta.CD (FIG. 4). At pH 7.5 the heptakis 6 amino
.beta.-cyclodextrin displays a third current level (not shown). It
causes dTMP to display two types of events with different
amplitudes, one of which is within the range of dGMP events, thus
leading to a loss of resolution. At pH 9.5, the nucleotide binding
events are no longer observed. The best peak separation is obtained
at pH 8.0.
Salt Dependence
[0131] The resolution between dXMPs and XMPs is better with KCl
than with NaCl or CsCl. 1M KCl yields better resolution than 2M
KCl. The use of KBr did not allow the identification of the
different nucleotides as each binding event led to a complete block
of the transient complex (M113R).sub.7/am.sub.7-.beta.CD.
Temperature Dependence
[0132] Lowering the temperature to 14.degree. C. or increasing it
to 50.degree. C. did not interfere with the detection of the
dXMP/XMP. However, it did not improve the resolution of the
amplitude histograms.
Other .alpha.-hemolysin Mutants
[0133] (M113N).sub.7 was seen to bind am.sub.7-.beta.CD but no
nucleotide detection was observed. (M113K).sub.7 and
(M113K/147K).sub.7 didn't yield detection whether am.sub.7-.beta.CD
was added or not. (M113K).sub.7 was tested in the same conditions.
In this case, the recording is very similar to that of FIG. 1. The
nucleotide binding was detected with (M113K).sub.7 mutant and
am.sub.7-.beta.CD but the peak separation between the nucleotides
was smaller than when (M113R).sub.7 was used.
Ribonucleotides 5' Monophosphate
[0134] XMP were successfully identified with this method, the
resolution between each base was inferior to that of dXMP with peak
separations smaller than 1 pA for U, A, and C. The all point
histogram current amplitudes appear in the same order as those of
dXMPs, with GMP displaying the lowest current (largest blocking),
followed by UMP, AMP, and CMP with the highest current amplitude
(smallest blocking). The optimal voltage for XMP identification was
found to be +110 mV at pH 8.0 .mu.M KCl.
Mechanism
[0135] The (M113R).sub.7/am.sub.7-.beta.CD transient complex has
also shown to bind and differentiate glucose phosphates (glucose 1P
and galactose 1P). It suggests a strong interaction between the
arginine ring on one side, the phosphate group and the amine ring
from the am.sub.7-.beta.CD on the other side. Unmodified
P-cyclodextrin does not yield any detection. Little difference is
observed between XMP and dXMP suggesting that the hydroxyl groups
do not play a large role in the binding
Sequence CWU 1
1
91882DNAStaphylococcus aureusCDS(1)..(879) 1gca gat tct gat att aat
att aaa acc ggt act aca gat att gga agc 48Ala Asp Ser Asp Ile Asn
Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15aat act aca gta aaa
aca ggt gat tta gtc act tat gat aaa gaa aat 96Asn Thr Thr Val Lys
Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn20 25 30ggc atg cac aaa
aaa gta ttt tat agt ttt atc gat gat aaa aat cac 144Gly Met His Lys
Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His35 40 45aat aaa aaa
ctg cta gtt att aga acg aaa ggt acc att gct ggt caa 192Asn Lys Lys
Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln50 55 60tat aga
gtt tat agc gaa gaa ggt gct aac aaa agt ggt tta gcc tgg 240Tyr Arg
Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp65 70 75
80cct tca gcc ttt aag gta cag ttg caa cta cct gat aat gaa gta gct
288Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val
Ala85 90 95caa ata tct gat tac tat cca aga aat tcg att gat aca aaa
gag tat 336Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys
Glu Tyr100 105 110atg agt act tta act tat gga ttc aac ggt aat gtt
act ggt gat gat 384Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val
Thr Gly Asp Asp115 120 125aca gga aaa att ggc ggc ctt att ggt gca
aat gtt tcg att ggt cat 432Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala
Asn Val Ser Ile Gly His130 135 140aca ctg aaa tat gtt caa cct gat
ttc aaa aca att tta gag agc cca 480Thr Leu Lys Tyr Val Gln Pro Asp
Phe Lys Thr Ile Leu Glu Ser Pro145 150 155 160act gat aaa aaa gta
ggc tgg aaa gtg ata ttt aac aat atg gtg aat 528Thr Asp Lys Lys Val
Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn165 170 175caa aat tgg
gga cca tat gat aga gat tct tgg aac ccg gta tat ggc 576Gln Asn Trp
Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly180 185 190aat
caa ctt ttc atg aaa act aga aat ggt tct atg aaa gca gca gat 624Asn
Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp195 200
205aac ttc ctt gat cct aac aaa gca agt tct cta tta tct tca ggg ttt
672Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly
Phe210 215 220tca cca gac ttc gct aca gtt att act atg gat aga aaa
gca tcc aaa 720Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys
Ala Ser Lys225 230 235 240caa caa aca aat ata gat gta ata tac gaa
cga gtt cgt gat gat tac 768Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu
Arg Val Arg Asp Asp Tyr245 250 255caa ttg cat tgg act tca aca aat
tgg aaa ggt acc aat act aaa gat 816Gln Leu His Trp Thr Ser Thr Asn
Trp Lys Gly Thr Asn Thr Lys Asp260 265 270aaa tgg aca gat cgt tct
tca gaa aga tat aaa atc gat tgg gaa aaa 864Lys Trp Thr Asp Arg Ser
Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys275 280 285gaa gaa atg aca
aat taa 882Glu Glu Met Thr Asn2902293PRTStaphylococcus aureus 2Ala
Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser1 5 10
15Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn20
25 30Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn
His35 40 45Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala
Gly Gln50 55 60Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly
Leu Ala Trp65 70 75 80Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro
Asp Asn Glu Val Ala85 90 95Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser
Ile Asp Thr Lys Glu Tyr100 105 110Met Ser Thr Leu Thr Tyr Gly Phe
Asn Gly Asn Val Thr Gly Asp Asp115 120 125Thr Gly Lys Ile Gly Gly
Leu Ile Gly Ala Asn Val Ser Ile Gly His130 135 140Thr Leu Lys Tyr
Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro145 150 155 160Thr
Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn165 170
175Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr
Gly180 185 190Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys
Ala Ala Asp195 200 205Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu
Leu Ser Ser Gly Phe210 215 220Ser Pro Asp Phe Ala Thr Val Ile Thr
Met Asp Arg Lys Ala Ser Lys225 230 235 240Gln Gln Thr Asn Ile Asp
Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr245 250 255Gln Leu His Trp
Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp260 265 270Lys Trp
Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys275 280
285Glu Glu Met Thr Asn29031300DNAArtificial sequenceM113H alpha
hemolysin mutant 3gttctgttta actttaagaa gggagatata catatgag cag att
ctg ata ttn acn 56Gln Ile Leu Ile Xaa Thr1 5tnn gcg acc ggt act aca
gat att gga agc aat act aca gta aaa aca 104Xaa Ala Thr Gly Thr Thr
Asp Ile Gly Ser Asn Thr Thr Val Lys Thr10 15 20ggt gat tta gtc act
tat gat aaa gaa aat ggc atg cac aaa aaa gta 152Gly Asp Leu Val Thr
Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val25 30 35ttt tat agt ttt
atc gat gat aaa aat cac aat aaa aaa ctg cta gtt 200Phe Tyr Ser Phe
Ile Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val40 45 50att aga aca
aaa ggt acc att gct ggt caa tat aga gtt tat agc gaa 248Ile Arg Thr
Lys Gly Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu55 60 65 70gaa
ggt gct aac aaa agt ggt tta gcc tgg cct tca gcc ttt aag gta 296Glu
Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val75 80
85cag ttg caa cta cct gat aat gaa gta gct caa ata tct gat tac tat
344Gln Leu Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr
Tyr90 95 100ccg cgg aat tcg att gat aca aaa gag tat cac agt acg tta
acg tac 392Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr His Ser Thr Leu
Thr Tyr105 110 115gga ttc aac ggt aac ctt act ggt gat gat act agt
aaa att gga ggc 440Gly Phe Asn Gly Asn Leu Thr Gly Asp Asp Thr Ser
Lys Ile Gly Gly120 125 130ctt att ggg gcc cag gtt tcc cta ggt cat
aca ctt aag tat gtt caa 488Leu Ile Gly Ala Gln Val Ser Leu Gly His
Thr Leu Lys Tyr Val Gln135 140 145 150cct gat ttc aaa aca att ctc
gag agc cca act gat aaa aaa gta ggc 536Pro Asp Phe Lys Thr Ile Leu
Glu Ser Pro Thr Asp Lys Lys Val Gly155 160 165tgg aaa gtg ata ttt
aac aat atg gtg aat caa aat tgg gga cca tac 584Trp Lys Val Ile Phe
Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr170 175 180gat cga gat
tct tgg aac ccg gta tat ggc aat caa ctt ttc atg aag 632Asp Arg Asp
Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys185 190 195act
aga aat ggt tct atg aaa gca gca gat aac ttc ctt gat cct aac 680Thr
Arg Asn Gly Ser Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn200 205
210aaa gca agt tcc cta tta tct tca ggg ttt tca cca gac ttc gct aca
728Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala
Thr215 220 225 230gtt att act atg gat aga aaa gca tcc aaa caa caa
aca aat ata gat 776Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln
Thr Asn Ile Asp235 240 245gta ata tac gaa cga gtt cgt gat gat tac
caa ttg cat tgg act tca 824Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr
Gln Leu His Trp Thr Ser250 255 260cca aat tgg aaa ggt acc aat act
aaa gat aaa tgg aca gat cgt tct 872Pro Asn Trp Lys Gly Thr Asn Thr
Lys Asp Lys Trp Thr Asp Arg Ser265 270 275tca gaa aga tat aaa atc
gat tgg gaa aaa gaa gaa atg aca aat taa 920Ser Glu Arg Tyr Lys Ile
Asp Trp Glu Lys Glu Glu Met Thr Asn280 285 290tgtaanttat ttgtacatgt
acaaataaat ataatttata actttagccg aagctggatc 980cggctgctac
naancccnaa ngnagctgan ttgnctgctg cccccctgac natactagca
1040naccccttgg gnccctaacg ggtctgnggg gtttttgctg aangngnact
tttccgnnan 1100tcnncccggn ccccccnggt gaaatccnaa nccccnaacn
ggngntgnta ncaantttan 1160tggnncntna ntttnnaaan cnnntaantt
ngnaancccc nttttncnan ggcnaannnn 1220nancctttna naaaaaancc
nnnggggggg tttcnntnnn annnccnttn aangggcccc 1280cnnggggnaa
nnntnggggn 13004293PRTArtificial sequencemisc_feature(5)..(5)The
'Xaa' at location 5 stands for Leu, or Phe. 4Gln Ile Leu Ile Xaa
Thr Xaa Ala Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15Asn Thr Thr Val
Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn20 25 30Gly Met His
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His35 40 45Asn Lys
Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln50 55 60Tyr
Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp65 70 75
80Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala85
90 95Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu
Tyr100 105 110His Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Leu Thr
Gly Asp Asp115 120 125Thr Ser Lys Ile Gly Gly Leu Ile Gly Ala Gln
Val Ser Leu Gly His130 135 140Thr Leu Lys Tyr Val Gln Pro Asp Phe
Lys Thr Ile Leu Glu Ser Pro145 150 155 160Thr Asp Lys Lys Val Gly
Trp Lys Val Ile Phe Asn Asn Met Val Asn165 170 175Gln Asn Trp Gly
Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly180 185 190Asn Gln
Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp195 200
205Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly
Phe210 215 220Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys
Ala Ser Lys225 230 235 240Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu
Arg Val Arg Asp Asp Tyr245 250 255Gln Leu His Trp Thr Ser Pro Asn
Trp Lys Gly Thr Asn Thr Lys Asp260 265 270Lys Trp Thr Asp Arg Ser
Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys275 280 285Glu Glu Met Thr
Asn29051300DNAArtificial sequenceM113K alpha hemolysin mutant
5gttctgttta actttaagaa gggagatata catatgag cag att ctg ata ttn acn
56Gln Ile Leu Ile Xaa Thr1 5tnn gcg acc ggt act aca gat att gga agc
aat act aca gta aaa aca 104Xaa Ala Thr Gly Thr Thr Asp Ile Gly Ser
Asn Thr Thr Val Lys Thr10 15 20ggt gat tta gtc act tat gat aaa gaa
aat ggc atg cac aaa aaa gta 152Gly Asp Leu Val Thr Tyr Asp Lys Glu
Asn Gly Met His Lys Lys Val25 30 35ttt tat agt ttt atc gat gat aaa
aat cac aat aaa aaa ctg cta gtt 200Phe Tyr Ser Phe Ile Asp Asp Lys
Asn His Asn Lys Lys Leu Leu Val40 45 50att aga aca aaa ggt acc att
gct ggt caa tat aga gtt tat agc gaa 248Ile Arg Thr Lys Gly Thr Ile
Ala Gly Gln Tyr Arg Val Tyr Ser Glu55 60 65 70gaa ggt gct aac aaa
agt ggt tta gcc tgg cct tca gcc ttt aag gta 296Glu Gly Ala Asn Lys
Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val75 80 85cag ttg caa cta
cct gat aat gaa gta gct caa ata tct gat tac tat 344Gln Leu Gln Leu
Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr90 95 100ccg cgg aat
tcg att gat aca aaa gag tat aaa agt acg tta acg tac 392Pro Arg Asn
Ser Ile Asp Thr Lys Glu Tyr Lys Ser Thr Leu Thr Tyr105 110 115gga
ttc aac ggt aac ctt act ggt gat gat act agt aaa att gga ggc 440Gly
Phe Asn Gly Asn Leu Thr Gly Asp Asp Thr Ser Lys Ile Gly Gly120 125
130ctt att ggg gcc cag gtt tcc cta ggt cat aca ctt aag tat gtt caa
488Leu Ile Gly Ala Gln Val Ser Leu Gly His Thr Leu Lys Tyr Val
Gln135 140 145 150cct gat ttc aaa aca att ctc gag agc cca act gat
aaa aaa gta ggc 536Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro Thr Asp
Lys Lys Val Gly155 160 165tgg aaa gtg ata ttt aac aat atg gtg aat
caa aat tgg gga cca tac 584Trp Lys Val Ile Phe Asn Asn Met Val Asn
Gln Asn Trp Gly Pro Tyr170 175 180gat cga gat tct tgg aac ccg gta
tat ggc aat caa ctt ttc atg aag 632Asp Arg Asp Ser Trp Asn Pro Val
Tyr Gly Asn Gln Leu Phe Met Lys185 190 195act aga aat ggt tct atg
aaa gca gca gat aac ttc ctt gat cct aac 680Thr Arg Asn Gly Ser Met
Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn200 205 210aaa gca agt tcc
cta tta tct tca ggg ttt tca cca gac ttc gct aca 728Lys Ala Ser Ser
Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr215 220 225 230gtt
att act atg gat aga aaa gca tcc aaa caa caa aca aat ata gat 776Val
Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp235 240
245gta ata tac gaa cga gtt cgt gat gat tac caa ttg cat tgg act tca
824Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln Leu His Trp Thr
Ser250 255 260cca aat tgg aaa ggt acc aat act aaa gat aaa tgg aca
gat cgt tct 872Pro Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys Trp Thr
Asp Arg Ser265 270 275tca gaa aga tat aaa atc gat tgg gaa aaa gaa
gaa atg aca aat taa 920Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys Glu
Glu Met Thr Asn280 285 290tgtaanttat ttgtacatgt acaaataaat
ataatttata actttagccg aagctggatc 980cggctgctac naancccnaa
ngnagctgan ttgnctgctg cccccctgac natactagca 1040naccccttgg
gnccctaacg ggtctgnggg gtttttgctg aangngnact tttccgnnan
1100tcnncccggn ccccccnggt gaaatccnaa nccccnaacn ggngntgnta
ncaantttan 1160tggnncntna ntttnnaaan cnnntaantt ngnaancccc
nttttncnan ggcnaannnn 1220nancctttna naaaaaancc nnnggggggg
tttcnntnnn annnccnttn aangggcccc 1280cnnggggnaa nnntnggggn
13006293PRTArtificial sequencemisc_feature(5)..(5)The 'Xaa' at
location 5 stands for Leu, or Phe. 6Gln Ile Leu Ile Xaa Thr Xaa Ala
Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15Asn Thr Thr Val Lys Thr Gly
Asp Leu Val Thr Tyr Asp Lys Glu Asn20 25 30Gly Met His Lys Lys Val
Phe Tyr Ser Phe Ile Asp Asp Lys Asn His35 40 45Asn Lys Lys Leu Leu
Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln50 55 60Tyr Arg Val Tyr
Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp65 70 75 80Pro Ser
Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala85 90 95Gln
Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr100 105
110Lys Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Leu Thr Gly Asp
Asp115 120 125Thr Ser Lys Ile Gly Gly Leu Ile Gly Ala Gln Val Ser
Leu Gly His130 135 140Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr
Ile Leu Glu Ser Pro145 150 155 160Thr Asp Lys Lys Val Gly Trp Lys
Val Ile Phe Asn Asn Met Val Asn165 170 175Gln Asn Trp Gly Pro Tyr
Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly180 185 190Asn Gln Leu Phe
Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp195 200 205Asn Phe
Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe210 215
220Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser
Lys225 230 235 240Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val
Arg Asp Asp Tyr245 250 255Gln Leu His Trp Thr Ser Pro Asn Trp Lys
Gly Thr Asn Thr Lys Asp260 265 270Lys Trp Thr Asp Arg Ser Ser Glu
Arg Tyr Lys Ile Asp Trp Glu Lys275 280 285Glu Glu Met Thr
Asn29071300DNAArtificial sequenceM113R alpha hemolysin mutant
7gttctgttta actttaagaa gggagatata catatgag cag att ctg ata ttn acn
56Gln Ile Leu Ile Xaa Thr1 5tnn gcg acc ggt act aca gat att gga agc
aat act aca gta aaa aca 104Xaa Ala Thr Gly Thr Thr Asp Ile Gly Ser
Asn Thr Thr Val Lys Thr10 15 20ggt gat tta gtc act tat gat aaa gaa
aat ggc atg cac aaa aaa gta 152Gly Asp Leu Val Thr Tyr Asp Lys Glu
Asn Gly Met His Lys Lys Val25 30 35ttt tat agt ttt atc gat gat aaa
aat cac aat aaa aaa ctg cta gtt 200Phe Tyr Ser Phe Ile Asp Asp Lys
Asn His Asn Lys Lys Leu Leu Val40 45 50att aga aca aaa ggt
acc att gct ggt caa tat aga gtt tat agc gaa 248Ile Arg Thr Lys Gly
Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu55 60 65 70gaa ggt gct
aac aaa agt ggt tta gcc tgg cct tca gcc ttt aag gta 296Glu Gly Ala
Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val75 80 85cag ttg
caa cta cct gat aat gaa gta gct caa ata tct gat tac tat 344Gln Leu
Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr90 95 100ccg
cgg aat tcg att gat aca aaa gag tat aga agt acg tta acg tac 392Pro
Arg Asn Ser Ile Asp Thr Lys Glu Tyr Arg Ser Thr Leu Thr Tyr105 110
115gga ttc aac ggt aac ctt act ggt gat gat act agt aaa att gga ggc
440Gly Phe Asn Gly Asn Leu Thr Gly Asp Asp Thr Ser Lys Ile Gly
Gly120 125 130ctt att ggg gcc cag gtt tcc cta ggt cat aca ctt aag
tat gtt caa 488Leu Ile Gly Ala Gln Val Ser Leu Gly His Thr Leu Lys
Tyr Val Gln135 140 145 150cct gat ttc aaa aca att ctc gag agc cca
act gat aaa aaa gta ggc 536Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro
Thr Asp Lys Lys Val Gly155 160 165tgg aaa gtg ata ttt aac aat atg
gtg aat caa aat tgg gga cca tac 584Trp Lys Val Ile Phe Asn Asn Met
Val Asn Gln Asn Trp Gly Pro Tyr170 175 180gat cga gat tct tgg aac
ccg gta tat ggc aat caa ctt ttc atg aag 632Asp Arg Asp Ser Trp Asn
Pro Val Tyr Gly Asn Gln Leu Phe Met Lys185 190 195act aga aat ggt
tct atg aaa gca gca gat aac ttc ctt gat cct aac 680Thr Arg Asn Gly
Ser Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn200 205 210aaa gca
agt tcc cta tta tct tca ggg ttt tca cca gac ttc gct aca 728Lys Ala
Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr215 220 225
230gtt att act atg gat aga aaa gca tcc aaa caa caa aca aat ata gat
776Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile
Asp235 240 245gta ata tac gaa cga gtt cgt gat gat tac caa ttg cat
tgg act tca 824Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln Leu His
Trp Thr Ser250 255 260cca aat tgg aaa ggt acc aat act aaa gat aaa
tgg aca gat cgt tct 872Pro Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys
Trp Thr Asp Arg Ser265 270 275tca gaa aga tat aaa atc gat tgg gaa
aaa gaa gaa atg aca aat taa 920Ser Glu Arg Tyr Lys Ile Asp Trp Glu
Lys Glu Glu Met Thr Asn280 285 290tgtaanttat ttgtacatgt acaaataaat
ataatttata actttagccg aagctggatc 980cggctgctac naancccnaa
ngnagctgan ttgnctgctg cccccctgac natactagca 1040naccccttgg
gnccctaacg ggtctgnggg gtttttgctg aangngnact tttccgnnan
1100tcnncccggn ccccccnggt gaaatccnaa nccccnaacn ggngntgnta
ncaantttan 1160tggnncntna ntttnnaaan cnnntaantt ngnaancccc
nttttncnan ggcnaannnn 1220nancctttna naaaaaancc nnnggggggg
tttcnntnnn annnccnttn aangggcccc 1280cnnggggnaa nnntnggggn
13008293PRTArtificial sequencemisc_feature(5)..(5)The 'Xaa' at
location 5 stands for Leu, or Phe. 8Gln Ile Leu Ile Xaa Thr Xaa Ala
Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15Asn Thr Thr Val Lys Thr Gly
Asp Leu Val Thr Tyr Asp Lys Glu Asn20 25 30Gly Met His Lys Lys Val
Phe Tyr Ser Phe Ile Asp Asp Lys Asn His35 40 45Asn Lys Lys Leu Leu
Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln50 55 60Tyr Arg Val Tyr
Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp65 70 75 80Pro Ser
Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala85 90 95Gln
Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr100 105
110Arg Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Leu Thr Gly Asp
Asp115 120 125Thr Ser Lys Ile Gly Gly Leu Ile Gly Ala Gln Val Ser
Leu Gly His130 135 140Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr
Ile Leu Glu Ser Pro145 150 155 160Thr Asp Lys Lys Val Gly Trp Lys
Val Ile Phe Asn Asn Met Val Asn165 170 175Gln Asn Trp Gly Pro Tyr
Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly180 185 190Asn Gln Leu Phe
Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp195 200 205Asn Phe
Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe210 215
220Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser
Lys225 230 235 240Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val
Arg Asp Asp Tyr245 250 255Gln Leu His Trp Thr Ser Pro Asn Trp Lys
Gly Thr Asn Thr Lys Asp260 265 270Lys Trp Thr Asp Arg Ser Ser Glu
Arg Tyr Lys Ile Asp Trp Glu Lys275 280 285Glu Glu Met Thr
Asn2909228PRTArtificial sequenceLambda exonuclease 9Ser His Met Thr
Pro Asp Ile Ile Leu Gln Arg Thr Gly Ile Asp Val1 5 10 15Arg Ala Val
Glu Gln Gly Asp Asp Ala Trp His Lys Leu Arg Leu Gly20 25 30Val Ile
Thr Ala Ser Glu Val His Asn Val Ile Ala Lys Pro Arg Ser35 40 45Gly
Lys Lys Trp Pro Asp Met Lys Met Ser Tyr Phe His Thr Leu Leu50 55
60Ala Glu Val Cys Thr Gly Val Ala Pro Glu Val Asn Ala Lys Ala Leu65
70 75 80Ala Trp Gly Lys Gln Tyr Glu Asn Asp Ala Arg Thr Leu Phe Glu
Phe85 90 95Thr Ser Gly Val Asn Val Thr Glu Ser Pro Ile Ile Tyr Arg
Asp Glu100 105 110Ser Met Arg Thr Ala Cys Ser Pro Asp Gly Leu Cys
Ser Asp Gly Asn115 120 125Gly Leu Glu Leu Lys Cys Pro Phe Thr Ser
Arg Asp Phe Met Lys Phe130 135 140Arg Leu Gly Gly Phe Glu Ala Ile
Lys Ser Ala Tyr Met Ala Gln Val145 150 155 160Gln Tyr Ser Met Trp
Val Thr Arg Lys Asn Ala Trp Tyr Phe Ala Asn165 170 175Tyr Asp Pro
Arg Met Lys Arg Glu Gly Leu His Tyr Val Val Ile Glu180 185 190Arg
Asp Glu Lys Tyr Met Ala Ser Phe Asp Glu Ile Val Pro Glu Phe195 200
205Ile Glu Lys Met Asp Glu Ala Leu Ala Glu Ile Gly Phe Val Phe
Gly210 215 220Glu Gln Trp Arg225
* * * * *
References